The focus of this research is to characterize the molecular mechanism of action of the yeast PUF proteins in the control of mRNA expression. The PUF proteins are members of a family of proteins containing an RNA binding motif consisting of 6-8 pumilio repeats. RNA binding by yeast and higher eukaryotic PUFs to mRNA 3' UTR sequences has been shown to be important to translational repression and accelerated mRNA degradation. PUF control of the mRNA deadenylation process has been shown to be the common requirement for both of these effects, although additional sites of PUF action have been suggested. In yeast each PUF protein can bind relatively distinct mRNAs, and the proteins encoded by each group of mRNAs are functionally related. These observations suggest that the PUFs play important post-transcriptional roles in controlling the expression of sets of proteins in the cell. We have identified several features of the PAB1- mRNP structure which affect the deadenylation process and these features may be sites through which PUF proteins act. We have shown that yeast PUFS can bind the CCR4-NOT deadenylase complex, suggesting that PUF3 retention of this complex can help accelerate deadenylation in vivo. We have also observed that PUFS can bind translation initiation factors, supporting a role of PUFS regulation of translation initiation as a means by which PUFS affects mRNA degradation In this proposal we will test several hypotheses concerning the mechanism of action of the yeast PUFS protein in terms of mRNA deadenylation. Using model mRNA controlled by PUFS (COX17), we will determine if the PUFS protein accelerates deadenylation by altering several different features of the mRNP structure involving the translation initiation complex, the translation termination factors, and PAB1. In addition, we will test the model that PUFS accelerates deadenylation by recruiting the CCR4-NOT deadenylase to the mRNA. Based on the large protein sizes of yeast PUFs, it is likely that they make multiple protein contacts and act through various means to ensure proper regulation of RNA expression. These experiments will utilize biochemical, genetic, and recombinant DNA techniques. Undergraduate students will be engaged in several aspects of this research project including that of mutating PUFS and translation initiation factors in order to localize the domains used in contacting each other. This proposal is relevant to public health by studying how protein expression is controlled. The characterization of the factors that regulate when and to what extent proteins are synthesized will illuminate the processes by which aberrant protein production leads to particular disease states. [unreadable] [unreadable] [unreadable]